EP1720053A1 - Microscope à balayage par laser - Google Patents

Microscope à balayage par laser Download PDF

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Publication number
EP1720053A1
EP1720053A1 EP06008983A EP06008983A EP1720053A1 EP 1720053 A1 EP1720053 A1 EP 1720053A1 EP 06008983 A EP06008983 A EP 06008983A EP 06008983 A EP06008983 A EP 06008983A EP 1720053 A1 EP1720053 A1 EP 1720053A1
Authority
EP
European Patent Office
Prior art keywords
sample
laser scanning
microscope
image
scanning microscope
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06008983A
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German (de)
English (en)
Inventor
Ralf Wolleschensky
Wolfgang Bathe
Frank Hecht
Ralf Engelmann
Jörg STEINERT
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Carl Zeiss Microscopy GmbH
Original Assignee
Carl Zeiss MicroImaging GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Carl Zeiss MicroImaging GmbH filed Critical Carl Zeiss MicroImaging GmbH
Publication of EP1720053A1 publication Critical patent/EP1720053A1/fr
Withdrawn legal-status Critical Current

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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/008—Details of detection or image processing, including general computer control

Definitions

  • frame rates of 25 frames / second or even significantly less (5 frames / second) are sufficient when setting the microscope parameters. It is essential that the scanner speed is not reduced as in the prior art (disadvantageous: changing the integration time and influencing the parameters for the image acquisition), but the pauses between the image recordings are increased.
  • the sample is less loaded. This can be done by making a separate adjustment for integration time and frame rate (pause time), for example via separate sliders.
  • FIG. 1 schematically shows a laser scanning microscope 1 which is essentially composed of five components: a radiation source module 2 which generates excitation radiation for laser scanning microscopy, a scanning module 3 which conditions the excitation radiation and deflects it appropriately for scanning via a sample, for simplification only schematically shown microscope module 4, which provided by the scan module Scanning radiation in a microscopic beam path directed to a sample, and a detector module 5, which receives and detects optical radiation from the sample.
  • the detector module 5 can, as shown in FIG. 1, be embodied spectrally with multiple channels.
  • the radiation source module 2 generates illumination radiation which is suitable for laser scanning microscopy, that is to say in particular radiation which can trigger fluorescence.
  • the radiation source module has a plurality of radiation sources for this purpose.
  • two lasers 6 and 7 are provided in the radiation source module 2, which are each followed by a light valve 8 and an attenuator 9 and couple their radiation via a coupling point 10 in an optical fiber 11.
  • the light valve 8 acts as a deflector with which a beam shutoff can be effected without having to switch off the operation of the laser in the laser unit 6 or 7 itself.
  • the light valve 8 is formed, for example, as AOTF, which deflects the laser beam before coupling into the optical fiber 11 in the direction of a light trap, not shown.
  • the laser unit 6 has three lasers B, C, D, whereas the laser unit 7 contains only one laser A.
  • the illustration is thus exemplary of a combination of single and multi-wavelength lasers, which are individually or jointly coupled to one or more fibers.
  • the coupling can also take place simultaneously via a plurality of fibers, the radiation of which is later mixed after passing through an adjustment optics by means of color combiner. It is thus possible to use a wide variety of wavelengths or ranges for the excitation radiation.
  • the coupled into the optical fiber 11 radiation is combined by means of slidable collimating optics 12 and 13 via beam combination mirror 14, 15 and changed in a beam shaping unit with respect to the beam profile.
  • the collimators 12, 13 ensure that the radiation supplied by the radiation source module 2 to the scanning module 3 is collimated into an infinite beam path. This is advantageously done with a single lens by displacement along the optical axis under control (not shown) central drive unit has a focusing function by the distance between the collimator 12, 13 and the respective end of the optical fiber is changeable.
  • the beam-shaping unit which will be explained in detail later, generates from the rotationally symmetric Gaussian-profiled laser beam, as it exists after the beam combination mirrors 14, 15, a line-shaped beam which is no longer rotationally symmetrical but is suitable in cross-section for producing a rectangularly illuminated field ,
  • This illumination beam which is also referred to as a line-shaped, serves as excitation radiation and is conducted to a scanner 18 via a main color splitter 17 and a zoom optical system to be described later.
  • the main color splitter will also be discussed later, it should merely be mentioned here that it has the function of separating sample radiation returning from the microscope module 4 from the excitation radiation.
  • the scanner 18 deflects the line-shaped beam in one or two axes, after which it is focused by a scanning objective 19 and a tube lens and an objective of the microscope module 4 into a focus 22 which lies in a preparation or in a sample.
  • the optical imaging is carried out so that the sample is illuminated in a focal line with excitation radiation.
  • the main color splitter 17 passes the fluorescence radiation lying in wavelength ranges other than the excitation radiation, so that it can be deflected via a deflection mirror 24 in the detector module 5 and then analyzed.
  • the detector module 5 has a plurality of spectral channels, ie, the fluorescent radiation coming from the deflection mirror 24 is split into two spectral channels in a secondary color splitter 25.
  • Each spectral channel has a slit diaphragm 26, which realizes a confocal or partially confocal imaging with respect to the sample 23 and whose size determines the depth of focus with which the fluorescence radiation can be detected.
  • the geometry of the slit diaphragm 26 thus determines the sectional plane within the (thick) preparation, from which fluorescence radiation is detected.
  • the slit diaphragm 26 is also arranged downstream of a block filter 27, which blocks unwanted excitation radiation that has entered the detector module 5.
  • the radiation separated in such a way and originating from a certain depth section is then analyzed by a suitable detector 28.
  • the second spectral detection channel is also constructed, which likewise comprises a slit diaphragm 26a, a block filter 27a and a detector 28a.
  • a confocal slot aperture in the detector module 5 is only exemplary.
  • a single-point scanner can be realized.
  • the slit diaphragms 26, 26a are then replaced by pinholes and the beam shaping unit can be dispensed with.
  • all such optics are designed rotationally symmetric for such a construction.
  • instead of a single-point sampling and detection in principle arbitrary multipoint arrangements, such as point clouds or Nipkow disk concepts, can be used. It is essential, however, that the detector 28 is spatially resolving, since a parallel detection of multiple sample points takes place during the passage of the scanner.
  • movable i.e., the movable, i.e. slidable collimators 12 and 13 present Gaussian beam over a mirror staircase in the form of the beam combination mirror 14, 16 combined and then converted in the illustrated construction with confocal slit diaphragm in a beam with rectangular beam cross-section.
  • a cylindrical telescope 37 is used in the beam-shaping unit, downstream of which is an aspherical unit 38, which is followed by a cylindrical optic 39.
  • the illumination assembly with the aspheric unit 38 may serve to uniformly fill a pupil between a tube lens and a lens.
  • the optical resolution of the lens can be fully utilized. This variant is therefore also useful in a single-point or multi-point scanning microscope system, z. In a line-scanning system (in the latter, in addition to the axis in which the sample is focused).
  • the z. B. linearly conditioned excitation radiation is directed to the main color divider 17.
  • This is in a preferred embodiment as a spectrally neutral splitter mirror according to the DE 10257237 A1 whose disclosure is fully incorporated herein.
  • the term "color divider” thus also includes non-spectral divider systems. Instead of the described spectrally independent color divider, it is also possible to use a homogeneous neutral divider (eg 50/50, 70/30, 80/20 or the like) or a dichroic divider. So that a selection is possible depending on the application, the main color divider is preferably provided with a mechanism which allows a simple change, for example by a corresponding Operarad containing individual, interchangeable divider.

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  • Physics & Mathematics (AREA)
  • Engineering & Computer Science (AREA)
  • Computer Vision & Pattern Recognition (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Microscoopes, Condenser (AREA)
  • Mechanical Optical Scanning Systems (AREA)
EP06008983A 2005-05-03 2006-04-29 Microscope à balayage par laser Withdrawn EP1720053A1 (fr)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
DE102005020540A DE102005020540A1 (de) 2005-05-03 2005-05-03 Laser-Scanning-Mikroskop

Publications (1)

Publication Number Publication Date
EP1720053A1 true EP1720053A1 (fr) 2006-11-08

Family

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Family Applications (1)

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EP06008983A Withdrawn EP1720053A1 (fr) 2005-05-03 2006-04-29 Microscope à balayage par laser

Country Status (4)

Country Link
US (1) US20060273261A1 (fr)
EP (1) EP1720053A1 (fr)
JP (2) JP5230857B2 (fr)
DE (1) DE102005020540A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3901683A1 (fr) * 2020-04-24 2021-10-27 Leica Microsystems CMS GmbH Procédé de commande d'imagerie d'un échantillon par un microscope et microscope correspondant
AU2022245975A1 (en) * 2021-03-25 2023-01-05 Illumina, Inc. Apparatus and methods for transmitting light

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0753880A1 (fr) * 1991-09-17 1997-01-15 Hitachi, Ltd. Microscope à balayage et procédé de mise en oeuvre d'un tel microscope à balayage
US20030142292A1 (en) * 2001-12-10 2003-07-31 Ralf Wolleschensky Arrangement for the optical capture of excited and/or back scattered light beam in a sample
US20040051040A1 (en) * 2001-08-29 2004-03-18 Osamu Nasu Method for measuring dimensions of sample and scanning electron microscope
EP1669789A1 (fr) * 2004-12-13 2006-06-14 Olympus Corporation Microscope à balayage laser

Family Cites Families (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH04159510A (ja) * 1990-10-24 1992-06-02 Hamamatsu Photonics Kk レーザ走査型観察装置
JPH095630A (ja) * 1995-06-15 1997-01-10 Nikon Corp 光走査方法
US6167173A (en) * 1997-01-27 2000-12-26 Carl Zeiss Jena Gmbh Laser scanning microscope
JPH11153758A (ja) * 1997-09-19 1999-06-08 Olympus Optical Co Ltd 走査型顕微鏡
JP2000066110A (ja) * 1998-08-18 2000-03-03 Nikon Corp 顕微鏡
JP2000066108A (ja) * 1998-08-18 2000-03-03 Nikon Corp 顕微鏡
JP4384290B2 (ja) * 1999-06-09 2009-12-16 オリンパス株式会社 走査型共焦点顕微鏡
JP4477170B2 (ja) * 1999-09-24 2010-06-09 オリンパス株式会社 走査型顕微鏡装置
JP2001166212A (ja) * 1999-12-07 2001-06-22 Olympus Optical Co Ltd 走査型顕微鏡装置
US6888148B2 (en) * 2001-12-10 2005-05-03 Carl Zeiss Jena Gmbh Arrangement for the optical capture of excited and /or back scattered light beam in a sample
US7196843B2 (en) * 2002-03-27 2007-03-27 Olympus Optical Co., Ltd. Confocal microscope apparatus
JP4564244B2 (ja) * 2003-06-26 2010-10-20 オリンパス株式会社 レーザ走査型顕微鏡、レーザ走査型顕微鏡の制御方法、及びプログラム
EP1498759B1 (fr) * 2003-07-15 2006-12-13 Yokogawa Electric Corporation Microscope confocale

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0753880A1 (fr) * 1991-09-17 1997-01-15 Hitachi, Ltd. Microscope à balayage et procédé de mise en oeuvre d'un tel microscope à balayage
US20040051040A1 (en) * 2001-08-29 2004-03-18 Osamu Nasu Method for measuring dimensions of sample and scanning electron microscope
US20030142292A1 (en) * 2001-12-10 2003-07-31 Ralf Wolleschensky Arrangement for the optical capture of excited and/or back scattered light beam in a sample
EP1669789A1 (fr) * 2004-12-13 2006-06-14 Olympus Corporation Microscope à balayage laser

Also Published As

Publication number Publication date
US20060273261A1 (en) 2006-12-07
JP2006313355A (ja) 2006-11-16
JP5230857B2 (ja) 2013-07-10
DE102005020540A1 (de) 2006-11-09
JP2012098755A (ja) 2012-05-24

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